[Isolation, identification and enzyme profiling of an alkaliphilic protease producing bacterium].

OBJECTIVE

To isolate and characterize alkaliphilic bacteria with protease activity.

METHODS

Protease-producing alkaliphiles were isolated by skim milk agar method. The morphological, biochemical and physiological characteristics, 16S rRNA gene sequence and DNA-DNA hybridization were determined to identify the taxonomic position of strain ZL223. Meanwhile, the enzyme characterization of the protease including optimal temperature and pH range, stability and oxidation resistance were tested by using casein hydrolysis test.

RESULTS

A new strain named ZL223 was isolated from Bange Lake in Tibet, China. Cells were Gram-positive, spore-forming, motile rods. Growth pH range was from 7.0 to 10.0, optimum at pH 9.0. Growth temperature range was from 15 degrees C to 45 degrees C, optimum at 37 degrees C. The G + C content of DNA was 40.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain belonged to the genus Bacillus and most closely related to Bacillus pseudofirmus OF4 with 98.6% sequence similarity. DNA-DNA hybridization analysis confirmed the close relationship of strain ZL223 with Bacillus pseudofirmu OF4 (86% relatedness). Strain ZL223 produced extracellular alkaline protease and its maximal enzyme activity was observed at 40 degrees C and pH 12.0.

CONCLUSION

Base on polyphasic taxonomy, strain ZL223 was a new member of Bacillus pseudofirmus. It produced alkaline protease which was worth further research.